PH responsive tertiary amine lipid compound and application thereof

Abstract

The invention provides a tertiary amine lipid compound with a structure as shown in a formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof, and an application of the tertiary amine lipid compound or the stereoisomer or the pharmaceutically acceptable salt thereof. The tertiary amine lipid compound is hydrophobic and electrically neutral under normal physiological pH, can be self-assembled with DSPE-PEG and other materials to form pH responsive broken material nanoparticles with PEG as a shell layer, and has weak interaction with a membrane and low toxicity to cells; under the condition of slightly acidic pH, part of tertiary amine can be protonized to form an amphiphilic structure consisting of a hydrophobic structural domain and a cationic structural domain, and the amphiphilic structure has strong interaction with cell membranes and organelle membranes and has strong membrane rupture activity, so that the cell membranes or lysosomal membranes can be efficiently and highly selectively destroyed, and tumor cells can be killed. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the technical field of materials and medicines, and in particular to a pH-responsive tertiary amine lipid compound and application thereof. Background Art

[0002] Malignant tumors have become a major threat to human life and health. Although current anti-tumor therapies such as chemotherapy, radiotherapy, and immunotherapy can inhibit tumor growth, prolong patient survival, and even completely cure tumors to a certain extent, they all face the problems of low specificity and large toxic side effects. For example, chemotherapy and radiotherapy can cause damage to normal tissues, and immune checkpoint therapy can cause immune-related toxic side effects.

[0003] Some cationic materials can kill tumor cells and pathogens such as bacteria by destroying cell membranes or organelle membranes. They have the advantages of broad-spectrum killing effects and are not easy to develop drug resistance. They have broad application prospects in the treatment of diseases such as tumors. For example, LTX-315 cationic peptides are used in tumor treatment through intratumoral injection, which can cause a large number of tumor cells to necrotize. It has achieved good results in melanoma, head and neck cancer, lymphoma, and breast cancer in combination with drugs such as ipilimumab and pembrolizumab. Related research is in Phase I clinical trials (https: / / clinicaltrials.gov / ). The study of LL-37 peptides for the treatment of melanoma through intratumoral injection is also in Phase I clinical research (https: / / clinicaltrials.gov / ). However, since this type of drug can not only efficiently destroy tumor cells and pathogens, but also has strong cytotoxicity to normal tissue cells, and peptide drugs are easily degraded by proteases, most drugs have stopped at clinical research, and most of the drugs currently used in clinical practice are local injection treatments.

[0004] Therefore, there is an urgent need to develop drugs that have no toxic side effects on normal tissues and cells and can specifically kill tumor cells for tumor treatment. Summary of the invention

[0005] Based on this, the present invention provides a class of pH-responsive tertiary amine lipid compounds, which are prepared by ring-opening reaction of primary amine or secondary amine with alkylene oxide, or by replacing the hydroxyl hydrogen with alkanoyl to obtain the corresponding derivatives. The present invention finds that partial modification of the hydroxyl group can reduce its cytotoxicity at pH 7.4, thereby improving its killing selectivity at normal physiological pH and tumor tissue pH. The tertiary amine lipid compound can be hydrophobic and electrically neutral at normal physiological pH, and can be self-assembled with amphiphilic auxiliary lipids such as DSPE-PEG to form lipid nanoparticles with PEG as the shell layer, which have low cytotoxicity; under slightly acidic pH conditions, it can be protonated to form an amphiphilic structure composed of a hydrophobic domain and a cationic domain, which has a strong interaction with the cell membrane or organelle membrane, thereby producing a strong cytotoxicity and specifically killing tumor cells.

[0006] The present invention includes the following technical solutions.

[0007] Application of a tertiary amine lipid compound having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof in the preparation of a pH-responsive membrane-breaking material:

[0008]

[0009] wherein each R1 is independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, alkenyl, alkynyl, aryl, aryl-substituted alkyl, cycloalkyl-substituted alkyl;

[0010] Each R2 is independently selected from: H, R1; wherein R is selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, aryl, aryl-substituted alkyl, cycloalkyl-substituted alkyl;

[0011] R3 and R4 are independently selected from: H, R9 substituted or unsubstituted alkyl, unsaturated chain hydrocarbon group, cycloalkyl, heterocyclic group, R8 substituted heterocyclic group, alkenyl, alkynyl, aryl, Or R3, R4 and the nitrogen atom connected thereto together form a 3-20 membered heterocyclic group which is substituted or unsubstituted with R8; and when R3 is directly connected to N, R3 is not H; when R4 is directly connected to N, R4 is not H;

[0012] Each R5 is independently selected from: alkylene, cycloalkyl, aryl;

[0013] Each R6 is independently selected from: alkyl, hydroxy substituted alkyl, unsaturated chain hydrocarbon, alkenyl, aryl, aryl substituted alkyl,

[0014] Each R7 is independently selected from: alkyl, alkyl substituted with hydroxyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, heterocyclic substituted with R8, alkenyl, alkynyl, aryl, alkyl substituted with aryl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8;

[0015] Each R8 is independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, alkyl substituted heterocyclic, alkenyl, alkynyl, aryl, aryl substituted alkyl, cycloalkyl substituted alkyl,

[0016] R9 is selected from the group consisting of: hydroxy, aryl, cycloalkyl, heterocyclyl, heterocyclyl substituted by R8, heteroaryl;

[0017] Each q and each q' is independently 0, 1 or 2;

[0018] a is 1, 2, 3, 4 or 5.

[0019] The tertiary amine lipid compound may have a structure as shown in the following formula (II):

[0020]

[0021] The tertiary amine lipid compound may also have a structure as shown in the following formula (III):

[0022]

[0023] R4 is selected from: R9 substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R8 substituted heterocyclic, alkenyl, alkynyl, aryl,

[0024] The tertiary amine lipid compound may also have a structure as shown in the following formula (IV):

[0025]

[0026] Where n is 1, 2, 3, 4 or 5;

[0027] R3 and R4 are independently selected from: C1-C 10 Alkyl, hydroxy substituted C1-C 10 Alkyl, C1-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, Or R3, R4 and the nitrogen atom to which they are connected together form a 3-8 membered heterocyclic group.

[0028] On the other hand, the present invention also provides a lipid nanoparticle, which is formed by self-assembly of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and amphiphilic molecules in an aqueous medium, wherein the amphiphilic molecules are: amphiphilic polyethylene glycol lipid molecules, or a combination of amphiphilic polyethylene glycol lipid molecules and phospholipids.

[0029] On the other hand, the present invention also provides a method for preparing the lipid nanoparticles, comprising the following steps:

[0030] dissolving the tertiary amine lipid compound or its stereoisomer or a pharmaceutically acceptable salt thereof in an organic solvent to obtain a tertiary amine lipid compound solution;

[0031] dissolving the amphiphilic molecule in an organic solvent to obtain an amphiphilic molecule solution;

[0032] Mixing the tertiary amine lipid compound solution and the amphiphilic molecule solution to obtain a mixed solution;

[0033] The lipid nanoparticles are obtained by adding an aqueous medium to the mixed solution and repeatedly blowing with a pipette; or, the mixed solution is slowly added dropwise to the aqueous medium under stirring and continued to stir to obtain the lipid nanoparticles.

[0034] On the other hand, the present invention also provides the use of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, or the lipid nanoparticle, including the following technical solutions.

[0035] The use of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt in the preparation of drugs for preventing and / or treating tumors.

[0036] The lipid nanoparticles are used in preparing drugs for preventing and / or treating tumors.

[0037] On the other hand, the present invention also provides a drug for preventing and / or treating tumors, which is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt described in the present invention, and / or the lipid nanoparticles.

[0038] The inventor's previous research shows that the protonatable tertiary amine membrane-breaking materials are hydrophobic and electrically neutral at normal physiological pH, and have low membrane lysis activity to cells and low toxicity to normal tissue cells due to the protonatable tertiary amine structure; and can be protonated in a slightly acidic environment, changing from electrically neutral to electrically positive, activating strong membrane lysis activity, and efficiently killing tumor cells, thereby achieving selective killing of tumor cells. On this basis, the inventors imagine that tertiary amine lipid compounds obtained by ring-opening primary or secondary amines with alkylene oxides may also have the same effect because they have similar structures. However, studies have found that this type of tertiary amine lipid compound has a poor selective killing effect on tumor cells in normal physiological environments and slightly acidic environments. On this basis, the present invention finds that partially modifying the hydroxyl groups of this type of tertiary amine lipid compound can reduce its cytotoxicity at normal tissue pH, and the partially modified tertiary amine lipid compound has a higher selective killing activity of cells at tumor acidity than the fully modified or unmodified tertiary amine lipid compound.

[0039] Based on the above technical solution, the present invention has the following beneficial effects:

[0040] The invention provides an acidity-activatable anti-tumor lipid tertiary amine small molecule compound, which consists of a tertiary amine head with convertible hydrophilic and hydrophobic properties and a tail containing a hydrophobic group. At normal physiological pH, the tertiary amine in the lipid material is hydrophobic and electrically neutral, and the tertiary amine head is in a hydrophobic conformation. Moreover, the lipid material can self-assemble with the assistance of auxiliary lipids such as DSPE-PEG to form acid-activatable anti-tumor lipid nanoparticles with PEG as the shell, and the interaction with the cell membrane is weak, so that it has the advantage of low toxicity to normal tissues when circulating in the body, and the PEG shell can improve the biocompatibility of the lipid material and prolong its blood circulation time; under the slightly acidic pH conditions of tumor tissue (a large number of studies have shown that tumor cells usually use glycolysis to produce more lactic acid and carbon dioxide, and because of the blockage of lymphatic reflux in solid tumor tissue, metabolic waste cannot be discharged normally, resulting in the peripheral environment pH of solid tumors being lower than the pH of normal body fluids, usually between 6.4 and 7.0), the tertiary amine part of the small molecule compound lipid material will be protonated, so that the nanoparticles form an amphiphilic structure composed of a hydrophobic domain and a cationic domain, so that it has a very strong interaction with the cell membrane or organelle membrane, so that it can kill tumor cells efficiently and selectively. The tertiary amine small molecule compound and / or the acidity-activatable antitumor lipid nanoparticles of the present invention can be used to prepare antitumor drugs, and have the advantages of good therapeutic effect, high selectivity and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1These are the nuclear magnetic resonance hydrogen spectra of C4OA25, C6OA25, C8OA25, C10OA25, C12OA25, C14OA25, C16OA25, and C18OA25.

[0042] Figure 2 These are the hydrogen nuclear magnetic resonance spectra of C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27.

[0043] Figure 3 These are the hydrogen nuclear magnetic resonance spectra of C14OEtA25, C14OBuA25, and C14OHexA25.

[0044] Figure 4 These are the hydrogen nuclear magnetic resonance spectra of C12OEtA25, C12OBuA25, and C12OHexA25.

[0045] Figure 5 These are the hydrogen nuclear magnetic resonance spectra of C12OEt2A25, C12OBu2A25, and C12OHex2A25.

[0046] Figure 6 These are the nuclear magnetic resonance hydrogen spectra of C12OOctA25, C10OHexA25, C10OOctA25, C10ODecA25, and C10ODodA25.

[0047] Figure 7 These are the nuclear magnetic resonance hydrogen spectra of C6OMyrA25, C6OPalA25, C6OSteA25, C8ODecA25, C8ODodA25, C8OMyrA25, and C8OPalA25.

[0048] Figure 8 This is a graph showing the relationship between cell activity and pH after the lipid nanoparticles of C6OA25, C8OA25, C10OA25, C12OA25, C14OA25, C16OA25, and C18OA25 prepared in Example 2 were incubated with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 hours.

[0049] Fig. 9 This is a graph showing the relationship between cell activity and pH after the lipid nanoparticles of C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27 prepared in Example 2 were incubated with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 hours.

[0050] Fig.10This is a graph showing the relationship between cell activity and pH after the lipid nanoparticles of C6OMyrA25, C6OPalA25, and C6OSetA25 prepared in Example 2 were co-incubated with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 hours.

[0051] Fig.11 This is a graph showing the relationship between cell activity and pH after the lipid nanoparticles of C8ODecA25, C8ODodA25, C8OMyrA25, and C8OPalA25 prepared in Example 2 were co-incubated with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 hours.

[0052] Fig.12 This is a graph showing the relationship between cell activity and pH after the lipid nanoparticles of C10OHexA25, C10OOctA25, C10ODecA25, and C10ODodA25 prepared in Example 2 were incubated with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 hours.

[0053] Fig.13 This is a graph showing the relationship between cell activity and pH after lipid nanoparticles of C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C12OEt2A25, C12OBu2A25, and C12OHex2A25 prepared in Example 2 were incubated with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 hours.

[0054] Fig.14 This is a graph showing the relationship between cell activity and pH after the lipid nanoparticles of C14OA25, C14OEtA25, and C14OBuA25 prepared in Example 2 were co-incubated with mouse pancreatic cancer Panc02 cells at pH 7.4 and 6.8 for 24 hours.

[0055] Fig.15 The particle size (a) and electric potential (b) of CnOA25 lipid nanoparticles prepared in Example 2 and the particle size (c) and electric potential (d) of CnOA27 lipid nanoparticles.

[0056] Fig.16 The particle size (ac) and potential (df) of CnORA25 lipid nanoparticles prepared in Example 2.

[0057] Fig.17 The particle size (ab) and electric potential (cd) of CnORA25 lipid nanoparticles prepared in Example 2.

[0058] Fig.18Changes in the protonation rate and pKa of lipid nanoparticles of C10OA25, C12OA25, C14OA25, C16OA25, C18OA25, C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27 prepared in Example 2 as a function of pH.

[0059] Fig.19 The protonation rate of lipid nanoparticles of C6OMyrA25, C6OPalA25, C6OSteA25, C8ODecA25, C8ODodA25, C8OMyrA25, C8OPalA25, C10OHexA25, C10OOctA25, C10ODecA25, C10ODodA25, C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C14OEtA25 and C14OBuA25 prepared in Example 2 changes with pH and their pKa.

[0060] Fig. 20 This is the liposome leakage rate of the lipid nanoparticles prepared in Example 2 at different pH values.

[0061] Fig.21 This is the hemolysis rate of the lipid nanoparticles prepared in Example 2.

[0062] Fig. 22 The toxicity of C14OEtA25 lipid nanoparticles on different tumor cells at the characteristic pH of tumor tissue.

[0063] Fig.23 Localization of C14OEtA25 lipid nanoparticles in tumor cells at the characteristic pH of tumor tissue.

[0064] Fig.24 Figure 2 shows the uptake of C14OEtA25 lipid nanoparticles by Panc02 tumor cells at pH 6.8 and pH 7.4.

[0065] Fig.25 The effects of endocytosis inhibitors (wortmannin, chlorpromazine hydrochloride, methyl-β-cyclodextrin, sodium azide) and temperature on the cytotoxicity of C14OEtA25 lipid nanoparticles at the characteristic pH of tumors.

[0066] Fig.26 This is the in vivo inhibitory effect of C14OEtA25 lipid nanoparticles on Panc02 tumors.

[0067] Fig. 27 This is the in vivo inhibitory effect of C14OEtA25 lipid nanoparticles on EO771 tumors.

[0068] Fig.28 This is the in vivo inhibitory effect of C14OEtA25 lipid nanoparticles on MC38 tumors. DETAILED DESCRIPTION

[0069] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0070] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0071] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0072] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0073] In the compounds described herein, when any variable (e.g., R1, R2, etc.) occurs more than once in any component, its definition at each occurrence is independent of the definition at every other occurrence. Likewise, combinations of substituents and variables are permitted as long as such combinations render the compound stable. Lines drawn from substituents into the ring system indicate that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, it means that such bonds are attached only to any suitable carbon atom of the adjacent ring. It is to be understood that one of ordinary skill in the art can select substituents and substitution patterns for the compounds of the present invention to provide chemically stable compounds that can be readily synthesized from readily available raw materials by techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it is to be understood that these groups may be on the same carbon atom or on different carbon atoms as long as the structure is stable. The phrase "optionally substituted with one or more substituents" is considered equivalent to the phrase "optionally substituted with at least one substituent" and in this case preferred embodiments will have 0-3 substituents.

[0074] As used herein, the term "alkyl" is intended to include branched and straight chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched arrangement. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0075] The term "unsaturated chain hydrocarbon group" refers to branched and straight-chain unsaturated aliphatic hydrocarbon groups having a specific number of carbon atoms, that is, non-cyclic unsaturated chain hydrocarbon groups, and the carbon chain contains one or more carbon-carbon double bonds, or contains carbon-carbon triple bonds, such as: CH2=CHCH2-, -(CH2)8(CH=CH)CH3, -(CH2)7CH=CH2, -(CH2)8CH=CH2, cis-9,12-octadecadienyl, etc.

[0076] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic, fused, spirocyclic, bridged or other cyclic substituent, wherein one or more ring atoms are selected from N, O or S(O)m (wherein m is an integer from 0 to 2) and the remaining ring atoms are carbon, for example: piperidinyl, pyrrolidinyl, etc.

[0077] The "acidity-activatable anti-tumor lipids" described in the present invention refer to lipids that can specifically activate anti-tumor activity to kill tumor cells under specific acidity conditions.

[0078] The "pH responsive membrane-breaking material" of the present invention refers to a material that has membrane-breaking activity only under specific acidity conditions. For example, it has membrane-breaking activity under conditions of pH less than 7, and has no membrane-breaking activity or very weak membrane-breaking activity under conditions of pH greater than 7. "Membrane-breaking activity" refers to binding, disturbing or directly destroying the cell membrane or organelle membrane by adhesion, insertion or membrane fusion.

[0079] The "tertiary amine lipid compound" or "lipid" or "small molecule compound" or "small molecule lipid compound" or "tertiary amine small molecule compound" described in the present invention all refer to compounds within the structural range shown by formula (I) to formula (V) of the present invention.

[0080] In one embodiment of the present invention, there is provided a use of a tertiary amine lipid compound having a structure represented by formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof in the preparation of a pH-responsive membrane-breaking material:

[0081]

[0082] wherein each R1 is independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, alkenyl, alkynyl, aryl, aryl-substituted alkyl, cycloalkyl-substituted alkyl;

[0083] Each R2 is independently selected from: H, R1; wherein R is selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, aryl, aryl-substituted alkyl, cycloalkyl-substituted alkyl;

[0084] R3 and R4 are independently selected from: H, R9 substituted or unsubstituted alkyl, unsaturated chain hydrocarbon group, cycloalkyl, heterocyclic group, R8 substituted heterocyclic group, alkenyl, alkynyl, aryl, Or R3, R4 and the nitrogen atom connected thereto together form a 3-20 membered heterocyclic group which is substituted or unsubstituted with R8; and when R3 is directly connected to N, R3 is not H; when R4 is directly connected to N, R4 is not H;

[0085] Each R5 is independently selected from: alkylene, cycloalkyl, aryl;

[0086] Each R6 is independently selected from: alkyl, hydroxy substituted alkyl, unsaturated chain hydrocarbon, alkenyl, aryl, aryl substituted alkyl,

[0087] Each R7 is independently selected from: alkyl, alkyl substituted with hydroxyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, heterocyclic substituted with R8, alkenyl, alkynyl, aryl, alkyl substituted with aryl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8;

[0088] Each R8 is independently selected from: alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, alkyl substituted heterocyclic, alkenyl, alkynyl, aryl, aryl substituted alkyl, cycloalkyl substituted alkyl,

[0089] R9 is selected from the group consisting of: hydroxy, aryl, cycloalkyl, heterocyclyl, heterocyclyl substituted by R8, heteroaryl;

[0090] Each q and each q' is independently 0, 1 or 2;

[0091] a is 1, 2, 3, 4 or 5.

[0092] In some preferred embodiments, the tertiary amine lipid compound has a structure shown in the following formula (II):

[0093]

[0094] In some preferred embodiments, the tertiary amine lipid compound has a structure shown in the following formula (III):

[0095]

[0096] R4 is selected from: R9 substituted or unsubstituted alkyl, unsaturated chain hydrocarbon, cycloalkyl, heterocyclic, R8 substituted heterocyclic, alkenyl, alkynyl, aryl,

[0097] In some preferred embodiments, R4 is selected from: C1-C 22 Alkyl, R9 substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, 3-22 membered heterocyclic group substituted by R8, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 22 Aryl,

[0098] In some preferred embodiments, R4 is selected from: C1-C 10 Alkyl, R9 substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl,

[0099] In some preferred embodiments, each R5 is independently selected from: C1-C 22 Alkylene, C3-C8 cycloalkyl, C6-C 14 Aryl.

[0100] In some preferred embodiments, each R5 is independently selected from: C1-C 10 Alkylene, C3-C8 cycloalkyl, phenyl, naphthyl, anthracenyl.

[0101] In some preferred embodiments, each R5 is independently selected from: C1-C6 alkylene, C3-C8 cycloalkyl.

[0102] In some preferred embodiments, each R5 is independently selected from: methylene, ethylene, propylene, butylene,

[0103]

[0104] In some preferred embodiments, each R6 is independently selected from: C1-C 22 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon, C2-C 22 Alkenyl, C6-C 14 Aryl, C6-C14 Aryl-substituted C1-C8 alkyl,

[0105] Each R7 is independently selected from: C1-C 22 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, 3-22 membered heterocyclic group substituted by R8, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8;

[0106] Each R8 is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, C1-C8 alkyl substituted 3-22 membered heterocyclic group, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl,

[0107] In some preferred embodiments, each R6 is independently selected from: C1-C 10 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon, C2-C 10 alkenyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthracenyl-substituted C1-C8 alkyl,

[0108] Each R7 is independently selected from: C1-C 10 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthracenyl-substituted C1-C8 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8;

[0109] Each R8 is independently selected from: C1-C 10 Alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, C1-C6 alkyl substituted 3-8 membered heterocyclic group, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthracenyl-substituted C1-C8 alkyl, C3-C8 cycloalkyl-substituted C1-C8 alkyl,

[0110] In some preferred embodiments, each R6 is independently selected from: C1-C6 alkyl; C1-C6 alkyl substituted with hydroxyl;

[0111] Each R7 is independently selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8;

[0112] Each R8 is independently selected from: C1-C6 alkyl, 5-8 membered heterocyclic group, 5-8 membered heterocyclic group substituted by C1-C6 alkyl, phenyl, C1-C3 alkyl substituted by phenyl,

[0113] In some preferred embodiments, R9 is selected from: hydroxyl, C6-C 14 Aryl, C3-C 22 Cycloalkyl, 3-22 membered heterocyclyl, 3-22 membered heterocyclyl substituted by R8, 5-10 membered heteroaryl.

[0114] In some preferred embodiments, R9 is selected from: hydroxyl, phenyl, naphthyl, anthracenyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, 3-8 membered heterocyclyl substituted by R8, 5-8 membered heteroaryl.

[0115] In some preferred embodiments, R4 is selected from: C1-C8 alkyl, C1-C8 alkyl substituted by R9, C3-C8 cycloalkyl, C1-C3 alkyl substituted by phenyl, C1-C3 alkyl substituted by naphthyl, C1-C3 alkyl substituted by anthracenyl, 3-8 membered heterocyclic group, 5-8 membered heterocyclic group substituted by R8, Substituted cyclohexyl;

[0116] R5 is selected from: C1-C4 alkylene;

[0117] R6 is selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl,

[0118] R7 is selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8, wherein the heteroatoms on the ring of the 5-8 membered heterocyclic group are 1 or 2 nitrogen atoms;

[0119] Each R8 is independently selected from: C1-C6 alkyl, phenyl, phenyl-substituted C1-C3 alkyl,

[0120] R9 is selected from the group consisting of: hydroxyl, C3-C8 cycloalkyl, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, 5-8 membered heteroaryl, and the heteroatom on the ring of the 3-8 membered heterocyclic group in R9 is 1 or 2 nitrogen atoms.

[0121] In some preferred embodiments, R4 is selected from: C4-C6 alkyl, butyl substituted by dimethylamino, butyl substituted by diethylamino, C1-C3 alkyl substituted by R9, C4-C8 cycloalkyl, benzyl, 5-8 membered heterocyclyl, 5-6 membered heterocyclyl substituted by R8, Substituted cyclohexyl;

[0122] R5 is selected from: C1-C3 alkylene;

[0123] R6 is selected from: C3-C4 alkyl;

[0124] R7 is selected from: C3-C4 alkyl;

[0125] Each R8 is independently selected from: C1-C6 alkyl, benzyl,

[0126] R9 is selected from:

[0127] In some embodiments, R4 is selected from: R9 substituted methyl, R9 substituted ethyl, R9 substituted propyl;

[0128] R9 is selected from:

[0129] In some embodiments, R4 is selected from:

[0130] R5 is selected from: C1-C3 alkylene;

[0131] R6, R7 and the nitrogen atom to which they are connected together form the following group:

[0132]

[0133]

[0134] In some preferred embodiments, the tertiary amine lipid compound has a structure as shown in the following formula (IV):

[0135]

[0136] Where n is 1, 2, 3, 4 or 5;

[0137] R3 and R4 are independently selected from: C1-C 10 Alkyl, hydroxy substituted C1-C 10 Alkyl, C1-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, Or R3, R4 and the nitrogen atom to which they are connected together form a 3-8 membered heterocyclic group.

[0138] In some preferred embodiments, R3 and R4 are independently selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, C1-C6 unsaturated chain hydrocarbon group, Or R3, R4 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group.

[0139] In some preferred embodiments, each R1 is independently selected from: C1-C 30 Alkyl, C2-C 30 Unsaturated chain hydrocarbon group, C3-C 30 Cycloalkyl, C2-C 30 Alkenyl, C2-C 30 Alkynyl, C6-C 30 Aryl, C6-C 14 Aryl substituted C1-C 12 Alkyl, C3-C 22 Cycloalkyl-substituted C1-C 12 alkyl.

[0140] In some preferred embodiments, each R1 is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 22 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl.

[0141] In some preferred embodiments, each R1 is independently selected from: C6-C 20 Alkyl, C6-C 20 Unsaturated chain hydrocarbon, C5-C 20 Cycloalkyl, C6-C 20 Alkenyl, C6-C 20 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C6 alkyl, naphthyl-substituted C1-C6 alkyl, anthracenyl-substituted C1-C6 alkyl, C5-C 20 Cycloalkyl-substituted C1-C6 alkyl.

[0142] In some preferred embodiments, each R1 is independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, isononyl, n-decyl, isodecanyl, undecyl, n-dodecyl, isododecyl, cyclododecyl, tridecyl, isotridecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexadecyl, heptadecyl, n-octadecyl, isooctadecyl, docosyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclododecylmethyl, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylmethyl, cyclobutylpropyl, cyclobutylbutyl, cyclopentyl methyl, cyclopentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, cyclohexylpentyl, cyclohexylhexyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, docosene, ethynyl, propynyl, butynyl, pentynyl, hexynyl, octynyl, decynyl, dodecynyl, tetradecynyl, hexadecynyl, octadecynyl, docosene, linolenyl, phenyl, naphthyl, anthracenyl, benzyl, phenethyl, phenpropyl, phenbutyl, phenhexyl, phenoctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, anthracenmethyl, anthracenethyl, anthracenpropyl, anthracenbutyl, anthracenhexyl, anthracenoctyl, cis-9,12-octadecadienyl, 9-octadecenyl.

[0143] In some preferred embodiments, each R1 is independently selected from: n-butyl, n-hexyl, n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

[0144] In some preferred embodiments, each R2 is independently selected from: H,

[0145] In some preferred embodiments, each R2 is not hydrogen at the same time, that is, at least one R2 is not hydrogen.

[0146] In some preferred embodiments, one R2 is The other R2 are all hydrogen.

[0147] In some preferred embodiments, the tertiary amine lipid compound is as shown in formula (V):

[0148]

[0149] Preferably, R2 is

[0150] In some preferred embodiments, each R is independently selected from: C1-C 30 Alkyl, C2-C 30 Unsaturated chain hydrocarbon group, C3-C 30 Cycloalkyl, C6-C 30 Aryl, C6-C 14 Aryl substituted C1-C 12 Alkyl, C3-C 22 Cycloalkyl-substituted C1-C 12 alkyl.

[0151] In some preferred embodiments, each R is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, C6-C 22 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl.

[0152] In some preferred embodiments, each R is independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, isononyl, n-decyl, isodecanyl, undecyl, n-dodecyl, isododecyl, cyclododecyl, tridecyl, isotridecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexadecyl, heptadecyl, n-octadecyl, isooctadecyl, docosyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclododecyl, methyl, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylmethyl, cyclobutylpropyl, cyclobutylbutyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, cyclohexylpentyl, cyclohexylhexyl, linolenyl, phenyl, naphthyl, anthracenyl, benzyl, phenethyl, phenpropyl, phenbutyl, phenhexyl, phenoctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, anthracenmethyl, anthracenethyl, anthracenpropyl, anthracenbutyl, anthracenhexyl, anthracenoctyl, cis-9,12-octadecadienyl, 9-octadecenyl.

[0153] In some preferred embodiments, the total number of carbon atoms in R1 and R2 is 8-32, preferably 22-28, and more preferably 24-26.

[0154] In some preferred embodiments, R3 and R4 are independently selected from: H, R9 substituted or unsubstituted C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, 3-22 membered heterocyclic group substituted by R8, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 22 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl, Or R3, R4 and the nitrogen atom to which they are connected together form a 3-10 membered heterocyclic ring which is substituted or unsubstituted with R8.

[0155] In some preferred embodiments, R3 and R4 are independently selected from: H, R9 substituted or unsubstituted C1-C 10 Alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, C2-C 10 Alkenyl, C2-C10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C6 alkyl, naphthyl-substituted C1-C6 alkyl, anthracenyl-substituted C1-C6 alkyl, C3-C8 cycloalkyl-substituted C1-C6 alkyl, Or R3, R4 and the nitrogen atom to which they are attached together form a 5-10 membered heterocyclic group which is substituted or unsubstituted with R8.

[0156] In some preferred embodiments, R3, R4 and the nitrogen atom to which they are connected together form the following group:

[0157]

[0158] In some preferred embodiments, the tertiary amine lipid compound is selected from the following compounds:

[0159]

[0160]

[0161] wherein each R1 is independently: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 2-phenylethyl or phenylbutyl;

[0162] Each R2 is independently selected from: H, acetyl, n-butyryl, n-hexanoyl, n-octanoyl, n-decanoyl, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl, n-octadecanoyl, 2-phenylacetyl or phenylbutyryl.

[0163] In some preferred embodiments, the total number of carbon atoms in R1 and R2 is 8-32, preferably 22-28, and more preferably 24-26.

[0164] In some preferred embodiments, at least one R2 is H, and R2 are not H at the same time.

[0165] In one embodiment of the present invention, a lipid nanoparticle is also provided, which is formed by self-assembly of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and amphiphilic molecules in an aqueous medium, wherein the amphiphilic molecules are: amphiphilic polyethylene glycol polymers, or a combination of amphiphilic polyethylene glycol polymers and phospholipids; the amphiphilic polyethylene glycol polymers are amphiphilic polyethylene glycol lipid molecules and / or polyethylene glycol-polyesters.

[0166] Wherein, the amphiphilic polyethylene glycol lipid molecule can be selected from but not limited to: polyethylene glycol-dipalmitoyl phosphatidylethanolamine, polyethylene glycol-distearoyl phosphatidylethanolamine, 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol, 2-(polyethylene glycol)-N,N-tetracosyl acetamide; the amphiphilic polyethylene glycol-polyester is selected from but not limited to: polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, polyethylene glycol-polylactic acid-glycolate; preferably, the amphiphilic polyethylene glycol The molecular weight of the polyethylene glycol in the amphiphilic polyethylene glycol-polyester is 500 g / mol to 20,000 g / mol, preferably 1,000 g / mol to 3,000 g / mol, and preferably 1,800 g / mol to 2,200 g / mol; preferably, the molecular weight of the polyester in the amphiphilic polyethylene glycol-polyester is 500 g / mol to 20,000 g / mol, preferably 1,000 g / mol to 3,000 g / mol, and preferably 1,800 g / mol to 2,200 g / mol.

[0167] The phospholipids can be selected from, but are not limited to, cholesterol, soybean lecithin, hydrogenated soybean lecithin, soybean lecithin, egg yolk lecithin, highly purified egg yolk lecithin, dilauroyl lecithin, dieucoyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine, dipalmitoyl lecithin, distearoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylglycerol, 1,2-palmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, lysophosphatidyl ethyl Alcoholamine, palmitoyl lysolecithin, myristoyl lysolecithin, stearoyl lysolecithin, dimyristoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, (2,3-dioleoyl-propyl)-trimethylammonium-chloride, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, 1,2-dioleyl-3-dimethylamino-propane, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 1,2-dioctadecenyloxy-3-methylammonium propane, (R)-2,3-bis(oleoyloxy)propyl hydrogen phosphate 2-[(2-aminoethyl)amino]ethyl ester.

[0168] In some preferred embodiments, the lipid nanoparticles are formed by self-assembly of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and amphiphilic polyethylene glycol lipid molecules in an aqueous medium; preferably, the amphiphilic polyethylene glycol lipid molecules are distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

[0169] In some preferred embodiments, the mass ratio of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt to the amphiphilic polyethylene glycol lipid molecule is 1:0.2-8, preferably 1:0.5-4, more preferably 1:0.5-2, more preferably 1:0.8-1.2; and most preferably 1:1.

[0170] In one embodiment of the present invention, a method for preparing the lipid nanoparticles is also provided, comprising the following steps:

[0171] dissolving the tertiary amine lipid compound or its stereoisomer or a pharmaceutically acceptable salt thereof in an organic solvent to obtain a tertiary amine lipid compound solution;

[0172] dissolving the amphiphilic molecule in an organic solvent to obtain an amphiphilic molecule solution;

[0173] Mixing the tertiary amine lipid compound solution and the amphiphilic molecule solution to obtain a mixed solution;

[0174] The lipid nanoparticles are obtained by adding an aqueous medium to the mixed solution and repeatedly blowing with a pipette; or, the mixed solution is slowly added dropwise to the aqueous medium under stirring and continued to stir to obtain the lipid nanoparticles.

[0175] In some preferred embodiments, the organic solvent is selected from ethanol, methanol, dimethyl sulfoxide, N,N-dimethylformamide or tetrahydrofuran.

[0176] In some preferred embodiments, the concentration of the tertiary amine lipid compound is 10 mg / mL-100 mg / mL, more preferably 40 mg / mL-60 mg / mL; the concentration of the amphiphilic molecule solution is 20 mg / mL-200 mg / mL, more preferably 50 mg / mL-100 mg / mL.

[0177] In some preferred embodiments, the volume ratio of the mixed solution to the aqueous medium is 1:1-20, more preferably 1:2-10.

[0178] In one embodiment of the present invention, there is also provided the use of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt in the preparation of a drug for preventing and / or treating tumors.

[0179] In one embodiment of the present invention, there is also provided the use of the lipid nanoparticles in the preparation of drugs for preventing and / or treating tumors.

[0180] In some preferred embodiments, the tumor is pancreatic cancer, melanoma, colorectal cancer, lung cancer, tongue squamous cell carcinoma, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, ovarian epithelial cancer, nasopharyngeal carcinoma, or brain cancer.

[0181] In one embodiment of the present invention, a drug for preventing and / or treating tumors is also provided, which is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt described in the present invention, and / or the lipid nanoparticles.

[0182] The compounds of formula (I)-(V) of the present invention can be used in combination with other known anti-tumor drugs. When administered in combination, the compounds of formula (I)-(V) and the known drugs can be independent administration units, or together form a combined administration unit; the compounds of formula (I)-(V) can be administered simultaneously or separately with other known anti-tumor drugs. When the compounds of formula (I)-(V) are taken simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs and the compounds of formula (I)-(V). Drug combination also includes taking the compounds of formula (I)-(V) and one or more other known drugs in overlapping time periods. When the compounds of formula (I)-(V) are used in combination with one or more other known drugs, the dosage of the compounds of formula (I)-(V) or the known drugs can be the same as the dosage of the single drug, or it can be lower than the dosage when they are taken alone.

[0183] The drugs or active ingredients that can be used in combination with the compounds of formula (I) to (V) include but are not limited to: immune checkpoint inhibitors, estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxins / cytostatics, antiproliferative agents, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitors, serine / Threonine protein inhibitors, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, MDM2 family protein inhibitors, IAP family protein inhibitors, STAT family protein inhibitors, PI3K inhibitors, AKT inhibitors, integrin blockers, interferon-α, interleukin-12, COX-2 inhibitors, p53, p53 activators, VEGF antibodies, EGF antibodies, etc.

[0184] In some embodiments, the drugs or active ingredients that can be used in combination with the compounds of formula (I)-formula (V) include but are not limited to: aldesleukin, alendronic acid, interferon, atrenoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglutethimide, amifostine, amrubicin, anacrine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, arnosin, 5-azacytidine, azathioprine, bacillus Calmette-Guérin or tice bacillus Calmette-Guérin, betadine, betamethasone acetate, betamethasone sodium phosphate preparation, bexarotene, bleomycin sulfate, bromourea, bortezomib, busulfan, calcitonin, alezosumab injection, capecitabine, carboplatin, casodex, ce fesone, cymoleukin, daunorubicin, chlorambucil, cisplatin, cladribine, cladribine, clodrophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin liposome, dexamethasone, dexamethasone phosphate, estradiol valerate, denileukin 2, depomet, deslorelin, delazosane, diethylstilbestrol, diflucan, docetaxel, doxorubicin, dronabinol, chin-166-chitosan complex, eligard, rasburicase, epirubicin hydrochloride, aprepitant, epirubicin, epoetin alfa, erythropoietin, epoetin, levamisole tablets, estradiol preparations, 17-β-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxyphosphoric acid, vanbifu, etoposide, farsole azole, tamoxifen preparations, filgrastim, finasteride, firapisti, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridine nucleoside monophosphate, 5-fluorouracil, fluoxymesterone, flutamide, formestan, 1-β-D-arabinofuranosylcytosine-5'-stearoyl phosphate, fotemustine, fulvestrant, gamma globulin, gemcitabine, gemtuzumab, imatinib mesylate, carmustine rice paper capsules, goserelin, granisiron hydrochloride, histrelin, and mexin, hydrocortisone, erythro-hydroxynonyl adenine, hydroxyurea, tentan isobetaminab, idarubicin, ifosfamide, interferon α, interferon-α2, interferon α-2A, interferon α-2B, interferon α-nl, interferon α-n3, interferon beta, interferon gamma-la, interleukin-2, intron A, Iressa, irinotecan, Kateri, lentinan sulfate, letrozole, leucovorin, leuprorelin acetate, levomizole, levofolinic acid calcium salt, levothyroxine sodium, levothyroxine sodium preparations, lomustine, lonidamine, dronabinol, nitrogen mustard, methylcobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogen, 6-mercaptopurine, mesna, methotrexate, methyl aminolevulinate, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, trilostane, liposomal doxorubicin citrate, nedaplatin, pegfilgrastim, oprelleukin, neupogen, nilutamide, tamoxifen,NSC-631570, recombinant human interleukin 1-β, octreotide, ondansetron hydrochloride, dehydrocortisone oral solution, oxaliplatin, paclitaxel, prednisone sodium phosphate preparation, pegaspargase, PEGASYS, pentostatin, lysin, pilocarpine hydrochloride, pilocarpine, plicamycin, porfimer sodium, prednimustine, stiprenisolone, prednisone, premarin, procarbazine, recombinant human erythropoietin, raltitrexed, libi, etidronate rhenium-186, rituximab, doxycycline-A, romotide, pilocarpine hydrochloride tablets, octreotide, sarmustine, semustine, sizoran, sobuzosine, sodium methylprednisolone, pafosic acid, stem cell therapy, streptozocin, strontium-89 chloride, levothyroxine sodium, tamoxifen Fen, tamsulosin, tasofenamin, tastolactone, taxotere, tesifazines, temozolomide, teniposide, testosterone propionate, methyltestosterone, thioguanine, thiotepa, thyroid stimulating hormone, tiludronic acid, topotecan, toremifene, tositumomab, trastuzumab, treosulfan, retinoic acid, methotrexate tablets, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, eufodine, uridine, valrubicin, vesirinone, vinblastine, vincristine, vinamide, vinorelbine, virulizine, dextromethorphan, statins ester, zofran, paclitaxel protein stabilized preparation, acolbifene, interferon r-lb, affinitak, aminopterin, arzoxifene, a soprisnil, atamestane, atrasentan, BAY43-9006, Avastin, CCI-779, CDC-501, Celebrex, cetuximab, clenatol, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, exitecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium-166DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, miproxifene, minocycline, MS- 209, liposomal MTP-PE, MX-6, nafarelin, nemorubicin, nevastatin, noratript, oblimersen, onco-TCS, osidem, paclitaxel polyglutamate, sodium bromoglutamate, PN-401, QS-21, quasi-sea, R-154, raloxifene, ranpirnase, 13-cis retinoic acid, satraplatin, ciocalcitol, T-138067, tarceva, paclitaxel docosahexaenoic acid, thymosin αl, gazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-lo7R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100 and zoledronic acid or a combination thereof.

[0185] The drug for preventing and / or treating tumors of the present invention can be used in non-human mammals or humans.

[0186] The pharmaceutically acceptable excipients used in the drug for preventing and / or treating tumors of the present invention refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity.

[0187] "Compatibility" herein means that the components in the composition can be blended with the active ingredient (tertiary amine lipid compounds represented by Formula I to Formula V) of the present invention and with each other without significantly reducing the efficacy of the active ingredient.

[0188] The pharmaceutically acceptable excipients used in the drug for preventing and / or treating tumors of the present invention include, but are not limited to, one or more of the following materials: at least one of solvents, excipients, fillers, extenders, binders, humectants, disintegrants, solubilizers, absorption accelerators, adsorbents, diluents, solubilizers, emulsifiers, lubricants, wetting agents, suspending agents, flavoring agents and fragrances.

[0189] Examples of pharmaceutically acceptable excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0190] There is no particular limitation on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

[0191] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

[0192] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients:

[0193] (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0194] (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic;

[0195] (c) humectants, for example, glycerin;

[0196] (d) disintegrating agents, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0197] (e) a buffering solvent, for example, paraffin;

[0198] (f) absorption accelerators, for example, quaternary ammonium compounds;

[0199] (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate;

[0200] (h) adsorbents, for example, kaolin; and

[0201] (i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffering agent.

[0202] The solid dosage forms can also be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They can contain opacifying agents, and the release of the active ingredient in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes.

[0203] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices.

[0204] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0205] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0206] The following are specific embodiments.

[0207] Example 1: Preparation of tertiary amine lipid compounds and their derivatives obtained by the reaction of alkylene oxide and amine molecules

[0208] In this embodiment, tertiary amine lipid compounds containing different types of hydrophobic groups and their derivatives of tertiary amine lipid compounds after acylation of monohydroxyl or dihydroxyl groups are prepared. These lipids are first synthesized by ring-opening reaction of alkylene oxides (C4O, C6O, C8O, C10O, C12O, C14O, C16O, C18O) containing different hydrophobic carbon chains with N-(2-aminoethyl)piperidine (A25) and N-(2-aminoethyl)homopiperidine (A27), wherein C4O, C6O, C8O, C10O, C12O, C14O, C16O, C18O are 1,2-butylene oxide (C4O), 1,2-hexylene oxide (C6O), 1,2-epoxy Octane (C8O), 1,2-epoxydecane (C10O), 1,2-epoxydodecane (C12O), 1,2-epoxytetradecane (C14O), 1,2-epoxyhexadecane (C16O), 1,2-epoxyoctadecane (C18O); the acylation of hydroxyl groups is synthesized by nucleophilic substitution reaction of the above-mentioned synthetic lipids with acyl chlorides of different hydrophobic carbon chains (acetyl chloride, n-butyryl chloride, n-hexanoyl chloride, n-octanoyl chloride, n-decanoyl chloride, n-dodecanoyl chloride, n-tetradecanoyl chloride, n-hexadecanoyl chloride, n-octadecanoyl chloride).

[0209] That is, in this embodiment, a series of tertiary amine lipid compounds represented by formula (A) and formula (B) are synthesized by ring-opening reaction of alkylene oxide with N-(2-aminoethyl)piperidine (A25) and N-(2-aminoethyl)homopiperidine (A27). The reaction formula is as follows:

[0210]

[0211]

[0212] Wherein, R1 is: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl.

[0213] Subsequently, a series of tertiary amine lipid compound derivatives represented by formula (C) and formula (D) were synthesized by nucleophilic substitution reaction of the above-synthesized lipid of formula (A) with acyl chlorides of different hydrophobic carbon chains. The reaction formula is as follows:

[0214]

[0215] Among them, R1 is: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl; R is: methyl, n-propyl, n-pentyl, n-heptyl, n-nonyl, n-undecyl, n-tridecyl, n-pentadecyl, n-heptadecyl.

[0216] The raw materials used in the two-step reaction (except for formula (A)) were purchased directly and used directly without purification.

[0217] The specific steps are as follows:

[0218] Preparation of tertiary amine lipid compounds with different types of hydrophobic groups:

[0219] 1 g (3.7 mmol-13.9 mmol) of alkylene oxide (C4O, C6O, C8O, C10O, C12O, C14O, C16O, C18O, respectively) was placed in a reaction container, and N-(2-aminoethyl)piperidine / N-(2-aminoethyl)homopiperidine was added at a molar ratio of alkylene oxide to N-(2-aminoethyl)piperidine of 2.4:1. After mixing evenly, the mixture was reacted at 90°C in a sealed state for two days. The product obtained by the reaction was purified by column chromatography and used.

[0220] Column chromatography: Silica gel was used as column filler, and dichloromethane: methanol = 1% to 8% was used as eluent for gradient elution to purify the synthesized tertiary amine lipid compound. The collected product was subjected to rotary evaporation and freeze-dried before use. Its NMR characterization is as follows Figure 1 , Figure 2 As shown, analysis showed that pure target products were obtained: C4OA25, C6OA25, C8OA25, C10OA25, C12OA25, C14OA25, C16OA25, C18OA25; C10OA27, C12OA27, C14OA27, C16OA27, C18OA27 and their corresponding structural formulas are shown below.

[0221]

[0222] The tertiary amine lipid compound prepared as above is abbreviated as CnOA25 / CnOA27, wherein CnO refers to the alkylene oxide in the raw material, A25 / A27 refers to N-(2-aminoethyl)piperidine / N-(2-aminoethyl)homopiperidine in the raw material, for example, C4OA25 refers to the tertiary amine lipid compound prepared by the above reaction of alkylene oxide (C4O) wherein R1 is ethyl and N-(2-aminoethyl)piperidine (A25).

[0223] Preparation of derivatives of tertiary amine lipid compounds modified with different groups:

[0224] Place 200 mg of CmOA25 in a reaction container and dissolve it with 5 mL of anhydrous dichloromethane, then add anhydrous triethylamine at a molar ratio of CmOA25 to anhydrous triethylamine of 1:2, and finally slowly drop the acid chloride into the container under an ice bath at a molar ratio of CmOA25 to acid chloride (acetyl chloride, n-butyryl chloride, n-hexanoyl chloride, n-octanoyl chloride, n-decanoyl chloride, n-dodecanoyl chloride, n-tetradecanoyl chloride, n-hexadecanoyl chloride, n-octadecanoyl chloride) of 1:1 or 1:2.5, after the addition is complete, react at room temperature overnight, and the product obtained by the reaction is purified by column chromatography to separate the product in which one hydroxyl hydrogen is replaced by an alkanoyl group or the product in which two hydroxyl hydrogens are replaced by an alkanoyl group and then used.

[0225] Column chromatography: Silica gel was used as column filler and dichloromethane:methanol = 4% was used as eluent to purify the derivatives of the tertiary amine lipid compound in which one hydroxyl hydrogen was replaced by an alkanoyl group and two hydroxyl hydrogens were replaced by an alkanoyl group. The collected product was used after rotary evaporation and freeze-drying. Its NMR characterization is as follows Figure 3 to Figure 7 As shown, analysis showed that pure target products C6OMyrA25, C6OPalA25, C6OSteA25, C8ODecA25, C8ODodA25, C8OMyrA25, C8OPalA25, C10OHexA25, C10OOctA25, C10ODecA25, C10ODodA25, C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C12OEt2A25, C12OBu2A25, C12OHex2A25, C14OEtA25, C14OBuA25, and C14OHexA25 were obtained, and their corresponding structural formulas are shown below.

[0226]

[0227]

[0228] The derivative of the tertiary amine lipid compound prepared above is referred to as CmOR x A25, wherein CmO refers to the alkylene oxide in the starting material, A25 refers to N-(2-aminoethyl)piperidine in the starting material, R x Refers to the modification group alkanoyl (when x is omitted, it means that one hydroxyl hydrogen in the tertiary amine lipid compound is replaced by alkanoyl (monosubstitution); when x is 2, it means that two hydroxyl hydrogens in the tertiary amine lipid compound are replaced by alkanoyl (disubstitution)). For example, C12OEtA25 refers to the derivative of the monosubstituted tertiary amine lipid compound prepared by the above reaction of C12OA25 and acetyl chloride.

[0229] Example 2 Preparation of lipid nanoparticles

[0230] Example 1 The synthesized tertiary amine lipid compound or its derivative has a hydrophobic carbon chain portion and a hydrophobic tertiary amine portion at pH 7.4, and is in the presence of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000 ) can self-assemble into nanoparticles in water with the assistance of

[0231] The specific operation is as follows: dissolve a tertiary amine lipid compound or its derivative in ethanol to obtain a lipid solution with a concentration of 50 mg / mL; 2000 Dissolve in ethanol to obtain DSPE-PEG with a concentration of 50 mg / mL 2000 Solution; take 10 μL lipid solution and 10 μL DSPE-PEG 2000 The solutions were mixed in a 1.5 mL centrifuge tube to obtain a mixed solution; 180 μL of sterile water was added thereto using a pipette, quickly added to the above mixed solution, and repeatedly blown with a pipette to obtain 2.5 mg / mL lipid nanoparticles.

[0232] Example 3 Cytotoxicity of lipid nanoparticles at characteristic pH of normal tissues and tumor tissues

[0233] In this example, the MTT (thiazolyl blue) method was used to evaluate the killing effect of the drug on tumor cells under pH 7.4 and pH 6.8, and the cytotoxicity of the series of lipid nanoparticles prepared by the method of Example 2 at the characteristic pH of normal tissues and tumor tissues was studied.

[0234] The specific experimental steps are as follows: Use 6 mol / L HCl solution to adjust the DMEM medium to pH = 6.8 and 7.4 for standby use, and add 25 mM HEPES to maintain pH stability. Mouse pancreatic cancer cell line Panc02 (purchased from ATCC) was cultured in DMEM medium containing 10% (v / v) fetal bovine serum. At pH 7.4 and pH 6.8, the lipid nanoparticles prepared in Example 2 were diluted to different concentrations (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) and cells (concentration of 1×10 5 The cells were incubated in a 96-well plate at 37°C in a carbon dioxide incubator for 24 hours, and then the original culture medium was discarded. MTT (10 μL 5 mg / mL MTT solution (dissolved in PBS) + 90 μL DMEM culture medium) solution was added. The cells were placed in a 37°C in a carbon dioxide incubator for 2 hours, and then the MTT solution was discarded. 100 μL dimethyl sulfoxide was added. The cells were placed on a shaker for 20 minutes, and then the OD 490 nm absorbance was tested using an enzyme marker and the cell survival rate was calculated. The cell survival rate was calculated as follows: Cell survival rate % = (OD 纳米颗粒组-OD 背景 )÷(OD PBS组 -OD 背景 )×100%, where OD 背景 For blank wells without cells and containing only MTT, OD PBS组 The control group contained cells and MTT and was treated with a drug concentration of 0 μg / mL.

[0235] The test results are as follows Figure 8-Figure 14 As shown: From the test results, it can be seen that the lipid nanoparticles prepared by the present invention show certain regularity in their cytotoxicity to tumor cells at pH = 7.4 and pH = 6.8. For lipid nanoparticles prepared from C8OA25, C10OA25, C12OA25, C14OA25, C16OA25, and C18OA25, as the hydrophobic alkyl chain increases, the cytotoxicity of tumor cells at pH = 7.4 and pH = 6.8 changes from being obviously toxic to having certain selective killing to being non-toxic, indicating that due to the increase in hydrophobicity, the cytotoxicity of lipid nanoparticles at pH = 7.4 and pH = 6.8 is weakened; the hydrophobic chain segment of C6OA25 is too short, resulting in that after it is prepared into lipid nanoparticles, at pH = 6.8, it is further hydrophilic due to protonation and dissolves in the solution and cannot interact with the cell membrane, resulting in lower cytotoxicity at pH = 6.8 ( Figure 8 ). For lipid nanoparticles prepared from C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27, as the hydrophobic alkyl chain increases, the cytotoxicity of tumor cells at pH = 7.4 and pH = 6.8 changes from being obviously toxic to having certain selective killing to a decrease in toxicity of both, indicating that due to the increase in hydrophobicity, the cytotoxicity of lipid nanoparticles at pH = 7.4 and pH = 6.8 is weakened ( Fig. 9). For C6OMyrA25, C6OPalA25, C6OSetA25, C8ODecA25, C8ODodA25, C8OMyrA25, C8OPalA25, C10OHexA25, C10OOctA25, C10ODecA25, C10ODodA25, C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C12OEt2A25, C12OBu2A25, C12OHex2A25, C14OEtA25, C14OB For lipid nanoparticles prepared by uA25, as the hydroxyl group is substituted and the hydrophobicity of the substituent group increases, its cytotoxicity at pH = 7.4 and pH = 6.8 is weakened, and the cell killing selectivity first increases and then decreases with the increase of the carbon chain length of the substituent group. At the same time, the monosubstituted lipid material in which one hydroxyl hydrogen is substituted by an alkanoyl group has better cell killing selectivity than the disubstituted lipid material in which two hydroxyl hydrogens are substituted by an alkanoyl group and the unsubstituted lipid material, that is, the monosubstituted lipid material in which one hydroxyl hydrogen is substituted by an alkanoyl group has lower cytotoxicity at pH = 7.4 and higher cytotoxicity at pH = 6.8 ( Figure 10-Figure 14 ). And according to Figure 8-Figure 14 In summary, when the total number of C atoms on the hydrophobic main chain and the hydrophobic side chain of the modified lipid material (i.e., removing the carbon atoms on A25) is 28-30, the resulting lipid nanoparticles have the strongest ability to selectively kill tumor cells Panc02 at pH = 6.8.

[0236] Example 4 Particle size potential of lipid nanoparticles at pH 7.4 and pH 6.8

[0237] Take 100 μL of a lipid nanoparticle solution with a concentration of 10 mg / mL, dilute it to 1 mL using 0.1× PBS solution with pH values ​​of 7.4 and 6.8, respectively, and then add it to the corresponding dedicated test cell, and use a nanoparticle size and Zeta potential meter to characterize the particle size and potential of the nanoparticles.

[0238] The test results are as follows Figure 15-17 As shown in the figure, the particle size of CnOA25 and CnOA27 nanoparticles ranges from 100 to 270 nm, and the potential ranges from 1.6 to 5.3 mV. As the hydrophobic carbon chain grows, the particle size of the nanoparticles increases, and there is no obvious pattern in the potential. In addition, the particle size and potential of some nanoparticles at pH 6.8 are slightly larger than those at pH 7.4 ( Fig.15 ); There is no obvious regularity in the particle size distribution of CnORA25 nanoparticles. From the perspective of the potential of the nanoparticles, the potential at pH 6.8 is slightly higher than that at pH 7.4 ( Fig.16 and Fig.17).

[0239] Example 5 Protonation degree of lipid nanoparticles at different pH

[0240] The lipid nanoparticles prepared in Example 2 were mixed with buffer solutions of different pH values ​​and TNS solutions, and 100 μL of the solution was added to a black flat-bottom 96-well plate. The fluorescence intensity was then measured using a full-wavelength microplate spectrophotometer, and the protonation degree and pKa were calculated by simulating the change curve. The specific method is as follows:

[0241] Buffer preparation: First, use ultrapure water to prepare a buffer solution containing 20mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 20mM 2-(N-morpholine)ethanesulfonic acid (MES), 20mM ammonium acetate, and 260mMNaCl. Then, take four 25mL portions of 2x buffer solution, add 2mL and 1mL of 1M HCl solution (No. 1 and No. 2) and 2mL and 1mL of 1MNaOH (No. 3 and No. 4) solution, respectively, and finally fill to 50mL with ultrapure water. Finally, use No. 1 and No. 4 solutions, No. 2 and No. 3 solutions, respectively, to prepare buffer solutions with pH values ​​of 6.5-10 and 3-6.5 by mutual titration (one interval of 0.5pH).

[0242] Measurement method: Take 980 μL of buffer solutions of different pH values ​​and add them into 1.5 mL EP tubes, then add 10 μL of 2.5 mg / mL lipid nanoparticles and 100 μM TNS aqueous solution in sequence. After mixing evenly, take 100 μL of the solution and add it into a black flat-bottom 96-well plate. Use a full-wavelength microplate spectrophotometer to measure the fluorescence intensity at excitation and emission wavelengths of 321 nm and 445 nm, respectively.

[0243] Calculation method: Use the sigmoid function for simulation analysis, define the maximum fluorescence intensity point in the curve as 100% protonation rate, and the minimum fluorescence intensity point in the curve as 0% protonation rate, and then normalize them to obtain a simulation curve graph. The pH value corresponding to the protonation rate of 50% in the graph is the pK value of this lipid nanoparticle. a .

[0244] The test results are as follows Fig.18 , Fig.19 As shown in the figure, it can be seen that the protonation rate of lipid nanoparticles will begin to decline in a certain range, but the range of change is different for different lipid nanoparticles. For example, C16OA25 has a sudden jump of 80% protonation rate change between pH 6.8 and pH 7.4, while C10OA25 has a slow decline of 80% protonation rate between pH 7.8 and pH 9.5. Fig.18The analysis revealed that the pK values ​​of C10OA25, C12OA25, C14OA25, C16OA25, and C18OA25 were a They are 8.27, 7.13, 7.21, 6.82, and 6.93 respectively; the pK values ​​of C10OA27, C12OA27, C14OA27, C16OA27, and C18OA27 are a They are 8.36, 7.79, 7.61, 7.07, and 7.78 respectively. From this, we can see that pK a The relationship with the length of the hydrophobic carbon chain is roughly as follows: as the length of the hydrophobic carbon chain increases, pK a It shows a trend of first decreasing and then increasing.

[0245] from Fig.19 The analysis can obtain the pK values ​​corresponding to C6OMyrA25, C6OPalA25, C6OSteA25, C8ODecA25, C8ODodA25, C8OMyrA25, C8OPalA25, C10OHexA25, C10OOctA25, C10ODecA25, C10ODodA25, C12OEtA25, C12OBuA25, C12OHexA25, C12OOctA25, C14OEtA25, and C14OBuA25. a They are: 6.27, 6.57, 6.34, 5.74, 6.14, 6.00, 5.94, 6.69, 5.84, 5.70, 5.86, 6.73, 6.14, 6.03, 5.83, 6.42, and 6.10.

[0246] Example 6 Lipid Nanoparticles Cause Liposome Leakage at Different pH

[0247] In this embodiment, the liposomes simulating the components of tumor cell membranes were used to conduct dye encapsulation leakage experiments, and the leakage of encapsulated dyes was used to illustrate the interaction between lipid nanoparticles and tumor cell membranes. Liposomes were prepared by thin film hydration. Given that tumor cells have high expression of phosphatidylserine on their cell membranes compared to normal cells, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS) and 1,2-di(cis-9-oleoyl)-sn-glycero-3-phosphocholine (DOPC) (mass ratio of 1:9) were used to simulate tumor cell membranes.

[0248] The preparation method is as follows: prepare buffer 1 (10mM Na2HPO4) and buffer 2 (a mixed solution of 10mM Na2HPO4 and 90mM NaCl) with deionized water, and adjust the solution pH to 7.0. Use buffer 1 to prepare 12.5mM, 8-amino-1,3,6-naphthalenetrisulfonic acid disodium salt (ANTS) and 45mmol / L 1,1'-[1,4-phenylenebis(methylene)]bis(4,4'-bipyridyl) dibromide (DPX) solution. Mix the two in a 1:1 (v / v) ratio. Take 2g of Sephadex G-50, soak it in buffer 2 for 7h, and fill it into the chromatography column with a height of about 10cm. Add 840 μL of chloroform solution as solvent to a clean 50 mL round-bottom flask, add 144 μL of 25 mg / mL DOPC (dissolved in chloroform) and 16 μL of 25 mg / mL POPS (dissolved in chloroform), blow evenly and use to prepare liposomes with DOPC:POPS=9:1 (w / w). Heat the water bath to 37°C, use a rotary evaporator to remove chloroform under reduced pressure (speed 200 rpm), gradually reduce pressure to 350 mbar with a gradient of 100 mbar, maintain for 30 min, then continue to reduce pressure to 100 mbar and maintain for 30 min, and finally use an oil pump to vacuum dry the residual chloroform to obtain a uniform lipid film. Add 2 mL of the above-prepared ANTS and DPX mixed solution to the 50 mL flask with a lipid film, and rotate and hydrate at normal pressure on a rotary evaporator for 1 hour at 37°C and 200 rpm. The obtained liposome solution was transferred into a 2 mL centrifuge tube. After repeated freezing and thawing 10 times using liquid nitrogen and a 37°C water bath, the solution was passed through a dextran gel column to separate the liposomes from free ANTS and DPX. Buffer 2 was used as the mobile phase and the liposome solution was collected in a 2 mL centrifuge tube. The obtained liposome solution was stored at room temperature in the dark and used within 48 hours.

[0249] The specific experimental method steps for determining the interaction between lipid nanoparticles and liposomes encapsulating fluorescent dyes are as follows: PBS is titrated with a 6M hydrochloric acid solution and a 6M sodium hydroxide aqueous solution to adjust its pH to obtain PBS solutions with pH values ​​of 6.8 and 7.4. Subsequently, 4 μL of a 5mg / mL lipid nanoparticle solution, 10 μL of a liposome solution, and 86 μL of a PBS buffer solution with different pH values ​​are added to a 96-well flat-bottom plate with a black bottom, wherein three replicate wells are set for each different pH value. A negative control group (a mixed solution of 90 μL of a 1× PBS solution with a pH value of 6.2 and 7.4 and 10 μL of liposomes) and a positive control group (a mixed solution of 90 μL of 2% TritonX-100 (prepared with water) and 10 μL of liposomes) are set up. After the configuration is completed, incubate at room temperature in the dark for 1 hour, and then use an ELISA instrument to measure the fluorescence intensity at an excitation wavelength of 360 nm and an emission wavelength of 530 nm. The fluorescence intensity of the experimental group is defined as I t The fluorescence intensity of the negative control group co-incubated with PBS and liposomes was defined as I n The fluorescence intensity of the positive control group co-incubated with 2% Triton X-100 and liposomes was defined as I p Finally, using the formula [(I t -I n ) / (I p -I n )]×100% to calculate the corresponding fluorescence leakage rate.

[0250] The test results are as follows Fig. 20 As shown in the figure, it can be seen that the lipid nanoparticles prepared by the present invention can cause the leakage of liposome-entrapped dyes, and the leakage rate caused at pH 6.8 is significantly higher than that at pH 7.4, indicating that the lipid nanoparticles of the present invention have the function of destroying the phospholipid bilayer membrane, and the membrane destruction ability at pH 6.8 is stronger than that at pH 7.4, indicating that the membrane destruction function of the lipid nanoparticles is pH responsive.

[0251] Example 7 Hemolytic Activity of Lipid Nanoparticles

[0252] This example tests the hemolytic activity of the lipid nanoparticles prepared in Example 2 and verified in Example 3 with pH-selective cytotoxicity.

[0253] The specific experimental steps are as follows: wash sheep blood twice with 1×PBS until the supernatant is clear, transparent and colorless after centrifugation, and then dilute the sheep blood with 1×PBS to 4%; take 16μL of lipid nanoparticles (1.25mg / mL) prepared with 1×PBS in a 96-well flat-bottom plate, add 34μL of 1×PBS, and then add 50μL of 4% sheep blood, and incubate in a 37°C incubator for 4h; centrifuge at 3000rpm for 10min, then aspirate 50μL of supernatant in another 96-well plate, and use a multifunctional microplate reader to detect the absorbance at 576nm; set up a PBS control group without lipid nanoparticles (50μL 1×PBS + 50μL 4% sheep blood), a nanoparticle control group containing only lipid nanoparticles, and a positive control group containing 0.2% triton X (50μL 0.2% triton X + 50μL 4% sheep blood). The hemolysis rate is calculated as follows: [(OD 实验组 -OD 对照组 )÷(OD triton -OD PBS )]×100%.

[0254] The test results are as follows Fig.21 As shown, it can be seen from the experimental results that the hemolytic activity of the lipid nanoparticles prepared by the present invention is relatively low, mostly below 5%, and the hemolytic activities of C6OPalA25 and C6OSteA25 are 11% and 7%, respectively.

[0255] Example 8 Maximum Tolerated Dose (MTD) Test of Lipid Nanoparticles in ICR Mice

[0256] In this example, ICR mice were used to test the survival rate of C14OEtA25 lipid nanoparticles at 200 mg / kg to evaluate their toxicity to mice.

[0257] The specific maximum tolerated dose (MTD) test method is as follows: C14OEtA25 lipid nanoparticles are injected into ICR mice through the tail vein at a dose of 200 mg / kg. Four ICR mice are tested in each group. After the injection, the survival rate of the mice is observed and counted, and the survival rate at a dose of 200 mg / kg is determined after 24 hours. The results show that at an injection dose of 200 mg / kg, the survival rate of C14OEtA25 is 100%, indicating that the toxicity of C14OEtA25 lipid nanoparticles is very low and the safety is very high.

[0258] Example 9 Toxicity of lipid nanoparticles to different tumor cells at pH characteristic of tumor tissue

[0259] In this example, the killing effect of the drug on different tumor cells at pH 6.8 was evaluated by the MTT (thiazolyl blue) method, and the toxicity of the C14OEtA25 lipid nanoparticles prepared by the method of Example 2 on different tumor cells at the characteristic pH of tumor tissue was studied.

[0260] The specific experimental steps are as follows: 6 mol / L HCl solution is used to adjust the DMEM medium to pH = 6.8 and 7.4 for standby use, and 25 mM HEPES is added to maintain pH stability. Mouse pancreatic cancer cell line Panc02, mouse breast cancer cell line EO771, human non-small cell lung cancer cell line A549, human tongue squamous cell carcinoma cell line Cal27, human tongue squamous cell carcinoma cisplatin-resistant cell line Cal27 / DDP, mouse breast cancer cell line 4T1, human cervical cancer cell line Hela, mouse melanoma cell line B16-F10, mouse breast cancer cell line EMT6, human liver cancer cell line HepG2, human breast cancer cell line MDA-MB-231, mouse colon cancer cell line MC38 are cultured in DMEM or 1640 medium containing 10% (v / v) fetal bovine serum. At pH 7.4 and pH 6.8, the C14OEtA25 lipid nanoparticles prepared in Example 2 were diluted to different concentrations (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL) using DMEM or 1640 medium and incubated with cells (concentration of 1×10 5 The cells were incubated in a 96-well plate at 37°C in a carbon dioxide incubator for 24 hours, and then the original culture medium was discarded. MTT (10 μL 5 mg / mL MTT solution (dissolved in PBS) + 90 μL DMEM culture medium) solution was added. The cells were placed in a 37°C in a carbon dioxide incubator for 2 hours, and then the MTT solution was discarded. 100 μL dimethyl sulfoxide was added. The cells were placed on a shaker for 20 minutes, and then the OD 490 nm absorbance was tested using an enzyme marker and the cell survival rate was calculated. The cell survival rate was calculated as follows: Cell survival rate % = (OD 纳米颗粒组 -OD 背景 )÷(OD PBS组 -OD 背景 )×100%, where OD 背景 For blank wells without cells and containing only MTT, OD PBS组 The control group contained cells and MTT and was treated with a drug concentration of 0 μg / mL.

[0261] The test results are as follows Fig. 22As shown: From the test results, it can be seen that the C14OEtA25 lipid nanoparticles prepared by the present invention have obvious cytotoxicity to various tumor cells at pH = 6.8. In addition to killing sensitive tumor cells, C14OEtA25 lipid nanoparticles can also kill drug-resistant tumor cells. The IC50 value of C14OEtA25 lipid nanoparticles for different tumor cells is between 30-70μg / mL.

[0262] Example 10 Localization of lipid nanoparticles in tumor cells at pH characteristic of tumor tissue

[0263] C14OEtA25 lipid nanoparticles (incorporated with DSPE-PEG-Cy5) were incubated with Panc02 cells in a pH 6.8 medium for 2 hours. The tumor cells were stained with the lysosomal dye Lysotracker and the nuclear dye Hoechst 33342. The confocal microscope was used to observe the fluorescence of the lipid nanoparticles and the lysosomes. The fluorescence of the lipid nanoparticles also overlapped with that of the lysosomes. Fig.23 ), indicating that C14OEtA25 lipid nanoparticles may kill tumor cells by acting on the cell membrane and lysosome of tumor cells.

[0264] Example 11 Uptake of lipid nanoparticles by tumor cells at pH 6.8 and pH 7.4

[0265] C14OEtA25 lipid nanoparticles (incorporated with DSPE-PEG-FITC) were incubated with Panc02 cells in medium at pH 6.8 and pH 7.4 for 1, 2, 4, and 17 hours. After removing the drug, the cells were digested and collected for flow cytometry. Before loading, 0.4% trypan blue solution was added to quench the fluorescence of extracellular FITC. The test found that at pH 6.8, cells would take up more lipid nanoparticles, and the difference in uptake increased with time ( Fig.24 ), indicating that C14OEtA25 lipid nanoparticles are protonated at the characteristic pH of tumor tissue and interact with tumor cells through charge interactions, thereby promoting the uptake of lipid nanoparticles.

[0266] Example 12 Effects of endocytosis inhibitors and temperature on the cytotoxicity of lipid nanoparticles at tumor-specific pH

[0267] C14OEtA25 lipid nanoparticles combined with different endocytosis inhibitors (wortmannin, chlorpromazine hydrochloride, methyl-β-cyclodextrin, sodium azide) were incubated with Panc02 cells in a medium with pH 6.8, and the cell activity was determined by the MTT method. Fig.25As shown, the three endocytosis inhibitors, wortmannin, chlorpromazine hydrochloride, and methyl-β-cyclodextrin, had no inhibitory effect on the cytotoxicity of C14OEtA25 lipid nanoparticles at different concentrations, while the cell activity after combined treatment with sodium azide was higher than that of cells treated with lipid nanoparticles alone, indicating that the entry of the lipid nanoparticles of the present invention into cells is not mediated by macropinocytosis, clathrin, caveolin, etc., but is energy-dependent.

[0268] C14OEtA25 lipid nanoparticles were incubated with Panc02 cells at 4°C or 37°C, and the cell activity was determined by MTT assay. Fig.25 As shown, the toxicity of lipid nanoparticles to tumor cells was significantly reduced in an environment of 4°C, further proving that the tumor-killing effect of the lipid nanoparticles of the present invention is energy-dependent.

[0269] Example 13 In vivo tumor inhibition experiment of lipid nanoparticles

[0270] The tumor inhibition effect of lipid nanoparticles was verified by in vivo experiments in C57 mouse pancreatic cancer subcutaneous tumor model (Panc02 cells), C57 mouse breast cancer tumor orthotopic model (EO771 cells), and C57 mouse colorectal cancer subcutaneous tumor model (MC38 cells). The operation is as follows:

[0271] Female mice (C57BL / 6, 6-8 weeks old) were injected subcutaneously on the back with 1×10 7 Panc02 cell suspension or MC38 cell suspension (100 μL) was used to construct a pancreatic cancer subcutaneous tumor model or a colorectal cancer subcutaneous tumor model. 1×10 7 EO771 cell suspension (100 μL) was added to construct an orthotopic breast cancer model. 3 (Tumor volume = length × width × width / 2), administered via tail vein, C14OEtA25 lipid nanoparticles were administered at a dose of 50 mg / kg. A negative control group was also set up, which was injected with an equal volume of PBS solution. The tumor size was measured using a vernier caliper, and the mouse weight was recorded.

[0272] In vivo tumor inhibition effect of Panc02 model Fig.26 As shown in the figure, according to the tumor growth curve, the tumor volume of the drug-treated group was inhibited to a certain extent compared with the PBS group. At the same time, after the mice underwent the entire treatment cycle, there was no difference in body weight between the experimental group and the PBS control group. In the EO771 tumor model ( Fig. 27), C14OEtA25 lipid nanoparticles had a significant inhibitory effect on tumor growth, and the tumors of mice in some treatment groups completely disappeared. At the same time, this treatment regimen had no significant effect on the weight of mice. In the MC38 tumor model ( Fig.28 ), C14OEtA25 lipid nanoparticles can also inhibit tumor growth. The tumor of one mouse in the treatment group completely disappeared. In addition, this treatment regimen had no significant effect on the weight of the mice.

[0273] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a tertiary amine lipid compound having a structure represented by formula (III) or a stereoisomer thereof or a pharmaceutically acceptable salt thereof in the preparation of a pH-responsive membrane-breaking material: in, Each R1 is independently selected from: C1-C 30 Alkyl, C2-C 30 Unsaturated chain hydrocarbon, C3-C 30 Cycloalkyl, C2-C 30 Alkenyl, C2-C 30 Alkynyl, C6-C 30 Aryl, C6-C 14 Aryl substituted C1-C 12 Alkyl, C3-C 22 Cycloalkyl-substituted C1-C 12 alkyl; Each R2 is independently selected from: H, R1; Wherein, R is selected from: C1-C 30 Alkyl, C2-C 30 Unsaturated chain hydrocarbon, C3-C 30 Cycloalkyl, C6-C 30 Aryl, C6-C 14 Aryl substituted C1-C 12 Alkyl, C3-C 22 Cycloalkyl-substituted C1-C 12 alkyl; R4 is selected from: C1-C 22 Alkyl, R9 substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, 3-22 membered heterocyclic group substituted by R8, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 22 Aryl, Each R5 is independently selected from: C1-C 22 Alkylene, C3-C8 cycloalkyl, C6-C 14 Aryl; Each R6 is independently selected from: C1-C 22 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon, C2-C 22 Alkenyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, Each R7 is independently selected from: C1-C 22 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, 3-22 membered heterocyclic group substituted by R8, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8; Each R8 is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, 3-22 membered heterocyclic group, C1-C8 alkyl substituted 3-22 membered heterocyclic group, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 14 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl, R9 is selected from: hydroxyl, C6-C 14 Aryl, C3-C 22 Cycloalkyl, 3-22 membered heterocyclyl, 3-22 membered heterocyclyl substituted by R8, 5-10 membered heteroaryl.

2. The use according to claim 1, characterized in that: R4 is selected from: C1-C 10 Alkyl, R9 substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl, 3. The use according to claim 2, characterized in that: Each R5 is independently selected from: C1-C 10 Alkylene, C3-C8 cycloalkyl, phenyl, naphthyl, anthracenyl; Preferably, each R5 is independently selected from: C1-C6 alkylene, C3-C8 cycloalkyl; Preferably, each R5 is independently selected from: methylene, ethylene, propylene, butylene, 4. The use according to claim 2, characterized in that: Each R6 is independently selected from: C1-C 10 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon, C2-C 10 alkenyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthracenyl-substituted C1-C8 alkyl, Each R7 is independently selected from: C1-C 10 Alkyl, hydroxy substituted C1-C8 alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthracenyl-substituted C1-C8 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8; Each R8 is independently selected from: C1-C 10 Alkyl, C2-C 10 Unsaturated chain hydrocarbon group, C3-C8 cycloalkyl group, 3-8 membered heterocyclic group, C1-C6 alkyl substituted 3-8 membered heterocyclic group, C2-C 10 Alkenyl, C2-C 10 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C8 alkyl, naphthyl-substituted C1-C8 alkyl, anthracenyl-substituted C1-C8 alkyl, C3-C8 cycloalkyl-substituted C1-C8 alkyl, R9 is selected from the group consisting of: hydroxy, phenyl, naphthyl, anthracenyl, C3-C8 cycloalkyl, 3-8 membered heterocyclyl, 3-8 membered heterocyclyl substituted by R8, and 5-8 membered heteroaryl.

5. The use according to claim 4, characterized in that: Each R6 is independently selected from: C1-C6 alkyl; C1-C6 alkyl substituted with hydroxyl; Each R7 is independently selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8; Each R8 is independently selected from: C1-C6 alkyl, 5-8 membered heterocyclic group, 5-8 membered heterocyclic group substituted by C1-C6 alkyl, phenyl, C1-C3 alkyl substituted by phenyl, 6. The use according to claim 5, characterized in that: R4 is selected from: C1-C8 alkyl, C1-C8 alkyl substituted by R9, C3-C8 cycloalkyl, C1-C3 alkyl substituted by phenyl, C1-C3 alkyl substituted by naphthyl, C1-C3 alkyl substituted by anthracenyl, 3-8 membered heterocyclic group, 5-8 membered heterocyclic group substituted by R8, Substituted cyclohexyl; R5 is selected from: C1-C4 alkylene; R6 is selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, R7 is selected from: C1-C6 alkyl, hydroxy-substituted C1-C6 alkyl, Or R6, R7 and the nitrogen atom to which they are connected together form a 5-8 membered heterocyclic group which is substituted or unsubstituted with R8, wherein the heteroatoms on the ring of the 5-8 membered heterocyclic group are 1 or 2 nitrogen atoms; Each R8 is independently selected from: C1-C6 alkyl, phenyl, phenyl-substituted C1-C3 alkyl, R9 is selected from the group consisting of: hydroxyl, C3-C8 cycloalkyl, 3-8 membered heterocyclic group, 3-8 membered heterocyclic group substituted by R8, 5-8 membered heteroaryl, and the heteroatom on the ring of the 3-8 membered heterocyclic group in R9 is 1 or 2 nitrogen atoms.

7. The use according to claim 6, characterized in that: R4 is selected from: C4-C6 alkyl, butyl substituted by dimethylamino, butyl substituted by diethylamino, C1-C3 alkyl substituted by R9, C4-C8 cycloalkyl, benzyl, 5-8 membered heterocyclic group, 5-6 membered heterocyclic group substituted by R8, Substituted cyclohexyl; R5 is selected from: C1-C3 alkylene; R6 is selected from: C3-C4 alkyl; R7 is selected from: C3-C4 alkyl; Each R8 is independently selected from: C1-C6 alkyl, benzyl, R9 is selected from:

8. The use according to claim 7, characterized in that: R4 is selected from: methyl substituted by R9, ethyl substituted by R9, propyl substituted by R9; R9 is selected from:

9. The use according to claim 6, characterized in that: R4 is selected from: R5 is selected from: C1-C3 alkylene; R6, R7 and the nitrogen atom to which they are connected together form the following group:

10. The use according to any one of claims 1 to 9, characterized in that: Each R1 is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, C2-C 22 Alkenyl, C2-C 22 Alkynyl, C6-C 22 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl; Preferably, each R1 is independently selected from: C6-C 20 Alkyl, C6-C 20 Unsaturated chain hydrocarbon, C5-C 20 Cycloalkyl, C6-C 20 Alkenyl, C6-C 20 Alkynyl, phenyl, naphthyl, anthracenyl, phenyl-substituted C1-C6 alkyl, naphthyl-substituted C1-C6 alkyl, anthracenyl-substituted C1-C6 alkyl, C5-C 20 Cycloalkyl-substituted C1-C6 alkyl. Preferably, each R1 is independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, isononyl, n-decyl, isodecanyl, undecyl, n-dodecyl, isododecyl, cyclododecyl, tridecyl, isotridecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexadecyl, heptadecyl, n-octadecyl, isooctadecyl, docosyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclododecylmethyl, cyclopropylmethyl, cyclopropylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylmethyl, cyclobutylpropyl, cyclobutylbutyl, cyclopentylmethyl, cyclo pentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, cyclohexylpentyl, cyclohexylhexyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, docoseneyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, octynyl, decynyl, dodecynyl, tetradecynyl, hexadecynyl, octadecynyl, docoseneyl, linolenyl, phenyl, naphthyl, anthracenyl, benzyl, phenethyl, phenpropyl, phenbutyl, phenhexyl, phenoctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, anthracenmethyl, anthracenethyl, anthracenpropyl, anthracenbutyl, anthracenhexyl, anthracenoctyl, cis-9,12-octadecadienyl, 9-octadecenyl; Preferably, each R1 is independently selected from: n-butyl, n-hexyl, n-octyl, isodecyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 9-octadecenyl, cis-9,12-octadecadienyl, 2-phenylethyl, or phenylbutyl.

11. The use according to any one of claims 1 to 9, characterized in that: The tertiary amine lipid compound is shown in formula (V): Preferably, R2 is 12. The use according to any one of claims 1 to 9, characterized in that: Each R is independently selected from: C1-C 22 Alkyl, C2-C 22 Unsaturated chain hydrocarbon group, C3-C 22 Cycloalkyl, C6-C 22 Aryl, C6-C 14 Aryl-substituted C1-C8 alkyl, C3-C 22 Cycloalkyl-substituted C1-C8 alkyl; Preferably, each R is independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, n-pentyl, isopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl, isononyl, n-decyl, isodecanyl, undecyl, n-dodecyl, isododecyl, cyclododecyl, tridecyl, isotridecyl, n-tetradecyl, pentadecyl, n-hexadecyl, isohexadecyl, heptadecyl, n-octadecyl, isooctadecyl, docosyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclododecylmethyl, cyclopropyl cyclopentyl, cyclopentylethyl, cyclopropylpropyl, cyclopropylbutyl, cyclobutylmethyl, cyclobutylmethyl, cyclobutylpropyl, cyclobutylbutyl, cyclopentylmethyl, cyclopentylethyl, cyclopentylpropyl, cyclopentylbutyl, cyclohexylmethyl, cyclohexylethyl, cyclohexylpropyl, cyclohexylbutyl, cyclohexylpentyl, cyclohexylhexyl, linolenyl, phenyl, naphthyl, anthracenyl, benzyl, phenethyl, phenpropyl, phenbutyl, phenhexyl, phenoctyl, naphthylmethyl, naphthylethyl, naphthylpropyl, naphthylbutyl, naphthylhexyl, naphthyloctyl, anthracenyl, anthracenyl, anthracenyl, anthracenylpropyl, anthracenbutyl, anthracenyl, anthracenyloctyl, cis-9,12-octadecadienyl, 9-octadecenyl.

13. The use according to any one of claims 1 to 9, characterized in that: The total number of carbon atoms in R1 and R2 is 8-32, preferably 22-28, more preferably 24-26.

14. The use according to claim 1, characterized in that: The tertiary amine lipid compound is selected from the following compounds: wherein each R1 is independently: ethyl, n-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, 2-phenylethyl or phenylbutyl; Each R2 is independently selected from: H, acetyl, n-butyryl, n-hexanoyl, n-octanoyl, n-decanoyl, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl, n-octadecanoyl, 2-phenylacetyl or phenylbutyryl; Preferably, the total number of carbon atoms in R1 and R2 is 8-32, preferably 22-28, more preferably 24-26; Preferably, at least one R2 is H, and R2 are not H at the same time.

15. The use according to claim 14, characterized in that: The tertiary amine lipid compound is selected from the following compounds:

16. The tertiary amine lipid compound according to any one of claims 1 to 15 or its stereoisomer or a pharmaceutically acceptable salt thereof.

17. A lipid nanoparticle, characterized in that: It is formed by self-assembly of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt described in any one of claims 1 to 15, and amphiphilic molecules in an aqueous medium, wherein the amphiphilic molecules are: amphiphilic polyethylene glycol polymers, or a combination of amphiphilic polyethylene glycol polymers and phospholipids; the amphiphilic polyethylene glycol polymers are amphiphilic polyethylene glycol lipid molecules and / or polyethylene glycol-polyesters.

18. The lipid nanoparticle according to claim 17, characterized in that The amphiphilic polyethylene glycol lipid molecule is selected from the group consisting of polyethylene glycol-dipalmitoylphosphatidylethanolamine, polyethylene glycol-distearoylphosphatidylethanolamine, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol, and 2-(polyethylene glycol)-N,N-cosylacetamide; the amphiphilic polyethylene glycol-polyester is selected from the group consisting of polyethylene glycol-polylactic acid, polyethylene glycol-polycaprolactone, and polyethylene glycol-polylactic acid-glycolate; Preferably, the molecular weight of the polyethylene glycol in the amphiphilic polyethylene glycol polymer is 500 g / mol to 20000 g / mol; Preferably, the molecular weight of the polyester in the amphiphilic polyethylene glycol-polyester is 500 g / mol to 20000 g / mol.

19. The lipid nanoparticle according to claim 17, characterized in that The phospholipids are selected from the group consisting of cholesterol, soybean lecithin, hydrogenated soybean lecithin, soybean lecithin, egg yolk lecithin, highly purified egg yolk lecithin, dilauroyl lecithin, dieucoyl phosphatidylcholine, dioleoyl lecithin, dimyristoyl lecithin, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine, dipalmitoyl lecithin, distearoyl phosphatidic acid, dipalmitoyl phosphatidic acid, dioleoyl phosphatidylglycerol, egg yolk phosphatidylglycerol, 1-palmitoyl-2-oleoyl phosphatidylglycerol, 1,2-palmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylserine, dioleoyl phosphatidylserine, lysophosphatidylethanolamine, Palmitoyl lysolecithin, myristoyl lysolecithin, stearoyl lysolecithin, dimyristoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, (2,3-dioleoyl-propyl)-trimethylammonium-chloride, 3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol hydrochloride, 1,2-dioleyl-3-dimethylamino-propane, 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, 1,2-dioctadecenyloxy-3-methylammonium propane, (R)-2,3-bis(oleoyloxy)propyl hydrogen phosphate 2-[(2-aminoethyl)amino]ethyl ester.

20. The lipid nanoparticle according to any one of claims 17 to 19, characterized in that The lipid nanoparticles are formed by self-assembly of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt, and amphiphilic polyethylene glycol lipid molecules in an aqueous medium; preferably, the amphiphilic polyethylene glycol lipid molecules are distearoyl phosphatidylethanolamine-polyethylene glycol 2000.

21. The lipid nanoparticle according to claim 20, characterized in that The mass ratio of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt to the amphiphilic polyethylene glycol lipid molecule is 1:0.2-8, preferably 1:0.5-4, more preferably 1:0.5-2, more preferably 1:0.8-1.2; and most preferably 1:

1.

22. A method for preparing the lipid nanoparticles according to any one of claims 17 to 21, characterized in that: The steps include: dissolving the tertiary amine lipid compound or its stereoisomer or a pharmaceutically acceptable salt thereof in an organic solvent to obtain a tertiary amine lipid compound solution; dissolving the amphiphilic molecule in an organic solvent to obtain an amphiphilic molecule solution; Mixing the tertiary amine lipid compound solution and the amphiphilic molecule solution to obtain a mixed solution; The lipid nanoparticles are obtained by adding an aqueous medium to the mixed solution and repeatedly blowing with a pipette; or, the mixed solution is slowly added dropwise to the aqueous medium under stirring and continued to stir to obtain the lipid nanoparticles.

23. The method for preparing lipid nanoparticles according to claim 22, characterized in that: The organic solvent is selected from ethanol, methanol, dimethyl sulfoxide, N,N-dimethylformamide or tetrahydrofuran; and / or, The concentration of the tertiary amine lipid compound is 10 mg / mL-100 mg / mL; and / or, the concentration of the amphiphilic molecule solution is 20 mg / mL-200 mg / mL; and / or, The volume ratio of the mixed solution to the aqueous medium is 1:1-20.

24. Use of the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt as claimed in any one of claims 1 to 15 in the preparation of a drug for preventing and / or treating tumors.

25. Use of the lipid nanoparticles according to any one of claims 17 to 21 in the preparation of a drug for preventing and / or treating tumors.

26. The use according to claim 24 or 25, characterized in that The tumor is pancreatic cancer, melanoma, colorectal cancer, lung cancer, tongue squamous cell carcinoma, cervical cancer, ovarian cancer, osteosarcoma, liver cancer, breast cancer, bladder cancer, ovarian epithelial cancer, nasopharyngeal carcinoma, and brain cancer.

27. A drug for preventing and / or treating tumors, characterized in that: The composition is prepared from active ingredients and pharmaceutically acceptable excipients, wherein the active ingredients include the tertiary amine lipid compound or its stereoisomer or its pharmaceutically acceptable salt according to any one of claims 1 to 15, and / or the lipid nanoparticles according to any one of claims 17 to 21.

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